(based on Olek et al., 1996, Schoenherr et al., 2003)
DNA methylation is a stable epigenetic mark, which can mediate gene silencing. Bisulfite sequencing allows for precise identification of methylated cytosines within DNA (Frommer et al. 1992). This method is based on different rate of chemical conversion of methylated and non-methylated cytosines to uracil where non-methylated cytosines are converted efficiently while methylated cytosines remain non-reactive. This method was further developed by embedding analyzed DNA into an agarose bead (Olek et al. 1996). The protocol presented here was further optimized for bisulfite sequencing of small samples where the starting material was a small number of cells (Fedoriw et al. 2004; Svoboda et al. 2004). The smallest amount of material from which several unique clones were recovered was 25 oocytes, which corresponds to 100 DNA molecules in the initial material (Svoboda et al. 2004). This protocol can be also used for analyzing up to 200ng purified genomic DNA in one sample.
Institute of Molecular Genetics, Academy of Sciences of the Czech Republic
Videnska 1083 - 142 20 Prague 4, Czech Rebulic